In vitro cell culture systems for the investigation of the morphogen Sonic hedgehog (Shh)

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Abstract

Sonic hedgehog (Shh) plays an important role in embryogenesis. It acts as a morphogen that diffuses to form a concentration gradient and works in a temporally and spatially controlled manner. Shh not only regulates organogenesis, such as formation of digits on limbs and organization of the brain, but also controls cell division of stem cells. Perturbations in Shh signaling have been implicated in developmental disorders and development of some cancers. However, owing to the dynamic in vivo environment and the high degree of complexity of the molecular processes and transports, there are challenges of investigating the cell-Shh interactions. Controlled immobilization of proteins like cell adhesion proteins, immunoglobulins, hormones, morphogens, including Shh, is an essential step for a large set of applications in biology, medicine and biotechnology ranging from developmental biology to tissue engineering and biosensing. Reliable attachment of biomolecules on solid man-made substrates opens routes for many new key experiments, allowing studies of the biomolecules in simplified and thus controllable conditions. By taking advantage of the immobilization models for studying complicated biological events, the objective of the current project was to organize Shh in vitro in a way mimicking the in vivo microenvironment, in which the morphogen is distributed with a concentration gradient extracellularly. The immobilization processes have to show selective binding chemistry, provide control of the positioning and orientation of Shh on the substrate, and need to allow access of Shh to its receptor. In the presented project, biologically active Shh protein variants were synthesised in vitro. Plasmids of Shh with different tags or linker groups for immobilization were made. Production of sufficient amount of Shh for immobilization and surface analysis was performed in the E. coli expression system; while the determination of biological activity of Shh was carried out with the proteins obtained from the HEK293 cell culture system. The C3H10T1/2 clone 8 and the Shh LIGHT II cells demonstrated that the in vitro expressed Shh proteins are biologically active. The Shh induced cellular responses are dosage dependent. Adsorption or immobilization of Shh on substrates by a variety of physical and chemical interactions was then developed and tested. These interactions include collagen matrix networking; nickel(II)/poly(6)histidine affinity; biotin/streptavidin affinity; and benzylguanine (BG)/SNAPTM covalent binding. Each of them exhibits different binding strength. Surface analysis illustrated that all of the applied systems are capable of immobilizing Shh by physisorption or chemical binding. Collagen permits motility of the adsorbed Shh. The biological activity of the protein is maintained to induce Shh signal specific cellular responses. The nickel(II)/poly(6)histidine and biotin/streptavidin systems facilitate Shh binding with high selectivity. The BG/SNAPTM approach promotes specific and irreversible immobilization of Shh. Further experiments are under progression for the establishment of a tailor-made platform for the in vitro investigation of the interaction of irreversibly bound Shh with the responsive cells.